A vast number of cellular processes depend on the cell's ability to change the shape of their membranes with astounding spatial and temporal accuracy. At a biochemical level, many of the players that participate in these processes are known. What remains unknown, however, is how ensembles of several proteins with often overlapping functions and interaction specificities reproducibly accomplish defined biological outcomes. The largest hurdle towards resolving the mysteries of membrane remodeling is to obtain structural information about the membrane-associated scaffolds that orchestrate every aspect of these processes from changing membrane curvature to membrane fission, and recruitment of the actin cytoskeleton. Overcoming this limitation, we have demonstrated that electron cryomicroscopy provides access to the architecture of membrane-associated scaffolds at resolutions sufficient for the generation of detailed mechanistic models. Exploiting this advance, the long term goals of this project are to understand how members of the BAR superfamily (bin-amphihpysin-rvs family) of proteins generate/stabilize/sense membrane curvature, and how these molecules can selectively recruit interaction partners from a pool of promiscuous multidomain proteins such as the fission GTPase dynamin and the cytoskeletal activator N-WASP. We will use a combination of electron cryomicroscopy, low angle scattering, electron paramagnetic resonance spectroscopy and in vitro biophysical structure-function experiments to pursue three specific aims: (1) we will expand the number of experimentally determined scaffold structures, which will teach us much about their design principles and how these designs contribute to membrane curvature generation and selection of interaction partners, (2) we will exploit what we already learned to test mechanistic models of early steps in scaffold assembly, which may provide vital clues how scaffold assembly is regulated and (3) we will lay the foundation for structural work on higher order macromolecular complexes that BAR-domain proteins form with two of their most important effectors: dynamin and N-WASP. Taken together, these studies will allow us to greatly advance understanding of one of the most fundamental aspects of life: the ability of cells to change the shape of their membranes with amazing spatial and temporal resolution. Understanding these processes will be essential to appreciate how imbalances and errors in membrane remodeling contribute to a broad spectrum of human diseases ranging from epilepsy to diabetes and cancer. PUBLIC HEALTH RELEVANCE: In order to live, cells must continuously change the shape of their membranes with high precision. How cells accomplish this complex task is poorly understood because we have almost no information about how the molecular machines that are responsible for these processes interact with the membranes they reshape. Overcoming this limitation, we established a procedure to visualize membrane-remodeling molecules as they are engaged to their targets. This - for the first time - allows us to closely examine how these molecules function, and how they interact with additional proteins whose recruitment results in a specific biological effect. Visualizing these interactions is key to understanding how errors in these processes can contribute to diseases as varied as epilepsy, diabetes and cancer.